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Chapter 12B: Manipulating DNA - Coggle Diagram
Chapter 12B: Manipulating DNA
Polymerase chain reaction: allows a target DNA region to be replicated into as many copies as desired
Reagents
Sequence-specific DNA primers flanking the region of interest: required for the DNA polymerase to start synthesis (complementary to sequence)
Nucleotides (ATGC): building blocks for the synthesis of new DNA strands
temperature-resistant DNA polymerase (taq polymerase): to replicate DNA
Steps: DNA doubles after each full cycle of PCR
1) Denaturation: heat solution so the individual strands of the template DNA separates (denatures)
2) Annealing: Cool down solution and the primers bind complemenetary to the 2 strands
3) Extension with Taq polymerase: solution is heated to optimal temperature for DNA polymerase and the polymerase elongates each primer with nucleotides
Gel Electrophoresis: determines the size of a DNA fragment (visualizes the results of PCR to see if the expected size matches the actual size)
DNA fragments are separated by size as they migrate through a gel in response to an electrical field
DNA samples are loaded into wells at one end of gel and a current is applied
DNA is NEGATIVELY charged so it moved from cathode (-) --> anode (+)
shorter molecules = faster, longer molecules = slower
separate/analyze DNA fragments based on size and charge
Recombinant DNA technology (cloning)
Restriction enzymes cut DNA at specific sequences, leaving "sticky ends"
sticky ends: there are single stranded sequences that can be paired with another complementary sequence
Requirements for creating a transgenic organism
Recombinant DNA that's capable of replicating/being transmitted during cell division
Cells (host) that are capable of accepting/maintaining the recombinant DNA
Method for identifying cells that have taken up the recombinant DNA
DNA from any source can be propagated in bacteria
Gene of interest from ANOTHER cell is inserted into plasmid (from bacteria)
Plasmid put back into the bacterial cell
The bacterial cell now contains the gene of interest
Cloning a gene in bacteria
1) Isolate a bacterial plasmid containing an antibiotic resistance gene (GENE OF INTEREST) and the DNA that you want to clone
2) Cut plasmid and foreign DNA with same restriction enzyme (so they have same overhangs)
3) Mix DNA and treat with DNA ligase to seal pieces together
4) Transform bacteria with plasmid
Plate bacteria on selective medium (containing an antibiotic) to identify those that contain the plasmid (those that are resistant to the antibiotic)
Cells that grow contain plasmid (because it is antibiotic resistant)
Making Transgenic Plants
most transgenic plants are created using Agrobacterium tumefaciens (causes tumors)
Get a Ti plasmid and remove the tumor-causing gene from it --> Cut the Ti (tumor inducing) plasmid with a restriction enzyme at a restriction site
Put in new DNA with the gene of interest
--> recombinant Ti plasmid
put recombinant Ti plasmid back into A. tumefaciens and infect plant cells
Regenerate transformed cells into plants
Plant with new trait
T-DNA is transferred INTO plant cell and integrates into chromosome (plant is now permanently transformed)
Making transgenic animals
methods
take embryo out of mouse --> put in retrovirus --> retrovirus is integrated into chromosome --> kid is screened for presence of transgene
inject desirable DNA in sperm --> embryo --> put embryo in mom --> kid is screened for presence of transgene
embryonic stem cells are treated with desirable gene DNA --> identify the targeted ES cells (the cells that took up this gene) --> inject ES cells into embryo --> put into mom --> kid is chimeric (have some cells that are transgenes, some not) --> breeding and selection to homozygosity of the transgene
Sequencing by synthesis
attach little sequences from organisms onto the slide so that the ENTIRE GENOME is on the slide
add nucleotides one by one (nucleotides are modified so that it blocks the addition of another nucleotide)
fluorescent colors assigned for each nucleotide
remove block, add nucleotides, look at colors
gives you sequence on the slide